Viability and survival test

Presentation

The survival test exploits the natural tendency of cells to die in culture and is therefore used to assess the neuroprotective properties of your compounds. This test can be combined with various cell types depending on the pathology you target to assess the neuroprotective effect of your compounds.

Validation data

Alzheimer disease (AD) is the most common cause of dementia. It is a neurodegenerative disease characterized by an extracellular accumulation of neurotoxic fibrillar amyloid beta peptide (Aβ). Intoxication of neuronal cultures with Aβ has been widely used to understand some of the mechanisms of cell death in AD and thus represents an instrumental in-vitro system to evaluate the efficiency of neuroprotective drug candidates.

13-day-old cortical neuron cultures are injured by an acute intoxication with glutamate.The neuroprotective effect of compounds is evaluated based on their ability to inhibit on this damage. In the pre-treatment protocol, test compound is added 24h before intoxication and in the post-treatment one, test compound is added immediately after intoxication. Neuronal death is assessed by measuring LDH activity in the media at 24h after glutamate exposure.

12-day-old cortical neuron cultures are injured by an acute intoxication with NMDA. The neuroprotective effect of compounds is evaluated based on their ability to inhibit on this damage. In the pre-treatment protocol, test compound is added 24h before intoxication and in the post-treatment one, test compound is added immediately after intoxication. Neuronal death is assessed by measuring LDH activity in the media at 48h after NMDA exposure.

Effect of MK-801 on neuronal death induced by glutamate (75 µM, 10 min). * p<0.05 compare the control group; # p<0.05 compare the intoxicated group

Evaluation of neuronal viability (Quantification of the presence of ATP by CellTiter Glo, Promega) in response to increasing doses of acute (10 min) glutamate intoxication on 13-day old culture of cortical neuron, in presence or absence of 1µM of MK-801.* p<0.05 compare the control group; # p<0.05 compare the corresponding glutamate group

Evaluation of neuronal toxicity (quantification of binding of cell impermeant cyanine dye to dsDNA by CellTox Green, Promega) in response to increasing doses of acute (10 min) glutamate intoxication on 13-day old culture of cortical neuron, in presence or absence of 1µM of MK-801.* p<0.05 compare the control group; # p<0.05 compare the corresponding glutamate group

Evaluation of neuronal toxicity (quantification of binding of cell impermeant cyanine dye to dsDNA by CellTox Green, Promega) in response to acute (10 min) glutamate intoxication on 13-day old culture of cortical neuron, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

Evaluation of neuronal viability (Quantification of the presence of ATP by CellTiter Glo, Promega) in response to acute (10 min) glutamate intoxication on 13-day old culture of cortical neuron, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

GLUTAMATE: 10,000 neurons per well

Release of LDH (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega) in response to increasing doses of acute (10 min) glutamate intoxication on 13-day old culture of cortical neuron, in presence or absence of 1µM of MK-801.* p<0.05 compare the control group; # p<0.05 compare the corresponding glutamate group

Effect of increased doses of acute (10min) glutamate on neurite network of 13-day old culture of cortical neuron, in presence or absence of 1µM of MK-801.* p<0.05 compare the control group; # p<0.05 compare the corresponding glutamate group

Release of LDH (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega) in response to acute (10 min) glutamate intoxication on 13-day old culture of cortical neuron, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

Effect of acute (10min) glutamate on neurite network of 13-day old culture of cortical neuron, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

Effect of acute (10min) glutamate on total area of neurite network of 13-day old culture of cortical neuron, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

Effect of acute (10min) glutamate on total length of neurite of 13-day old culture of cortical neuron, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

NMDA testing

Release of LDH (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega) in response to acute (75µM, 10 min) NMDA intoxication on 12-day old culture of cortical neuron at density of 10000 cells/well, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

Evaluation of neuronal toxicity (quantification of binding of cell impermeant cyanine dye to dsDNA by CellTox Green, Promega) in response to acute (75µM, 10 min) NMDA intoxication on 12-day old culture of cortical neuron at density of 35000 cells/well, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

Evaluation of neuronal viability (Quantification of the presence of ATP by CellTiter Glo, Promega) in response to acute (75µM, 10 min) NMDA intoxication on 12-day old culture of cortical neuron at density of 35000 cells/well, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

Effect of acute (75µM, 10min) NMDA on neurite network of 12-day old culture of cortical neuron at density of 10000 cells/well, in presence or absence of increasing doses of MK-801.* p<0.05 compare the corresponding glutamate group

References :

Parkinson's disease (PD) is a neurodegenerative disorder with motor symptoms caused by the loss of dopaminergic (DA) neurons in the striatum. 6-hydroxydopamine (6-OHDA) is a neurotoxin widely used in lab animals to produce striatal dopamine depletion.MPP+
is the active in-vivo metabolite of MPTP causing selective degeneration of nigrostriatal dopaminergic (DA) neurons in the PD. Similarly to the in-vivo situation, MPP+ induces a selective death of DA in primary cultures of rat mesencephalic neurons. Thus, MPP+ intoxication of mesencephalic neuronal culture appears as a relevant and a cost-effective alternative to the in-vivo experiments for the evaluation of new test compounds with potential beneficial effect on PD.